Ostrich Purin

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IN VITRO PROPAGATION OF THE OSTRICH FERN (Matteuccia struthiopteris)BRIAN W, DYKEMAN and BRUCE G. CUMMING

Plant Industry Branch, New Brunswick Department of Agriculture, Fredericton,N.B. E3B 5HI; and Department of Biology, Univeristy of New Brunswick, Fred-ericton, N.B. E3B 6EI. Received Il Jan. 1985, accepted )6 Apr. 1985DyreveN, B. W. eNo CurravrNc, B. G. 1985. In vitro propagation of the ostrichfern(Matteuccia struthiopteris). Can. J. Plant Sci. 65: 1025-1032.Methods were developed for the successful in vitro propagation of ostrich fern (Mat-teuccia struthiopteris (L.) Todaro) clones utilizing shoot tips derived by forcinglateral buds on the rhizome. Maximum shoot proliferation was attained with 6-furfurylaminopurine (kinetin) at 1.0 mg/L with half-strength Murashige and Skoog(MS) inorganic salts and sucrose, agar, NaH,PO., adenine sulphate, i-inositol andthiamine.HCl at 30 000, 4000, 85, 40, 100, 0.4 mglL, respectively. Excellent frondand root development was achieved with half-strength MS salts and sucrose, agar,i-inositol and thiamine.HCl at 7500, 4000, 100 and 0.4 mg/L, respectively. Themethods developed were satisfactory for a cross section of clones. Morphogenesisin vitro was dependent on medium osmotic potential.Key words: Matteuccia struthiopteris, in vitro propagation, tissue culture, mor-phogenesis, fern (ostrich)[Propagation in vitro de la matteuccie fougdre-ir-l'autruche (Matteuccia stuthiop-teris) .lTitre abr6g6: Propagation in vitro de la fougdre-d-l'autruche.Nous avons 6labor6 des m6thodes pour la propagation in vitro de clones de la mat-teuccie fougdre-d-l'auftvche (Matteuccin struthiopteris (L.) Todaro) d I'aide d'ex-tr6mit6s de tiges obtenues par forgage des bourgeons lat6raux des rhizomes. Nousavons obtenu une prolif6ration maximale avec 1,0 mg/L de furfurylamino six purine(kin6tine), une dilution de 50Vo de sels inorganiques de Murashige et Skoog (MS)et un m6lange de sucrose, d'agar, de NaH,PO., de sulfate d'addnine, de i-inositolet de thiamine.HCl ir raison de 30 000, 4000, 85, 40, 100 et 0,4 mg/L respective-ment. Par ailleurs, nous avons obtenu un excellent diieveloppement des frondes etdes racines avec des sels MS dilu6s d 50o/c et un melange de sucrose, d'agar, dei-inositol et de thiamine.HCl i raison de 7500, 4000, 100 et0,4 mglL respective-ment. Les mdthodes mises au point donnaient des r6sultats satisfaisants avec touteune gamme de clones. La morphog6ndse in vitro d6pendait du potentiel osmotiquedu milieu utilis6.Mots cl6s: Matteuccia struthiopteris,, propagation in vitro, culture de tissus,morpnogenese

The croziers of the ostrich fern. Matteucciastruthiopteris (L.) Todaro, have been har-vested as a spring food delicacy by the in-habitants of eastern North America for sev-eral centuries. The largest harvest ofcroziers or fiddleheads takes place in NewBrunswick and Maine where over 400 000Can. J. Plant Sci.65: 1025-1032 (Oct. 1985)

kg are picked annually from the wild. Pres-ent demand for fiddlehead greens is fargreater than available supply. Therefore,clonal selection and crop management re-search have been initiated with the objec-tive of developing a cultivated productionsystem for the species. This also necessi-tates the development of methods for as-exual propagation of superior clones.t025

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t026 CANADIAN JOURNAL OF PLANT SCIENCEThe natural asexual reDroductive svstemin the species is through the producrion ofrhizomes. New rhizomes arise from de-tached meristems (Wardlaw 1943) whichare laid down at regular intervals along therhizome and erect shoot (crown). Thisnormally results in the production of one tofour new rhizomes per crown per year. Ifrhizomes are detached from the crown andare sectioned into lengths of 5 cm, for ex-ample, the detached meristems are acti-vated, resulting in the development of newlateral shoots. The number of new olantswhich can be derived from this o.o..du...however. is inadequate for commercialneeds. For this reason coupled with the vervsuccessful in vitro systems developed ior

other ferns (Hughes l98l), effort was di-rected towards developing an in vitro prop-agation system. This is the subject of thefollowing study.MATERIALS AND METHODS

Explant Establishment in VitroVarious parts of the sporophytic plant werescreened as sources of explant material for cul-ture. Shoots produced from detached meristemson the rhizome were the only successful explantsand these were used as the initial source of ex-plants for all studies. Rhizomes were sectionedinto 2-cm pieces, placed in plastic bags andmaintained at 25'C in low light intensiry (100-500 lx). When the lateral shoots were 5-10 mmlong (6-8 wk), 5-mm shoot tips were removed,surface sterilized tn 10o/a commercial bleach(0.67o NaOCI) and0.l%o C5-28 1-3 wetting agent(Aerosol 22, American Cyanamid) for 10 min,and then rinsed twice in sterile deionized water.Shoots were then recut to l-2 mm and one ex-plant was placed in each 30-mL ointment jarcontaining l5 mL of medium.From preliminary studies a basal medium forestablishment was developed which containedMurashige and Skoog (MS) salts (Murashige andSkoog 1962) at 0.5 x strength and the followingin milligrams per litre: thiamine.HCl, 0.4; mon-obasic sodium phosphate, 85; adenine sulphate,40; i-inositol, 100; sucrose, 30 000; tissue cul-ture (TC) agar (K.C. Biologicals), 8000; 6-fur-furylamino purine (kinetin), 1.0; and naphthal-enaecetic acid (NAA), 1.0. All studies reported

here utilized shoots that were obtained by in vi-tro proliferation on the above medium.In Vitro Shoot MultiplicationTen shoots, each ca. 8 rng in weight, were placedon 50 mL of medium in a 300-mL ointment iar.Jars were covered with a double layer of siranfilm and maintained in continuous light (1000 lxfrom cool white lamps) at 25-r loC. Four rep-lications per treatment of four jars (40 shoots)each were used. Data were recorded after 8 wkin culture and included fresh weight, dry weight,number of shoots, average shoot weight, thenumber of fronds and roots per culture. Analysisof variance and an LSD test were carried out onall data.Three cytokinins: kinetin, N6-benzylaminopurine (BA) and N6 isopentenylamino purine(2iP) and three auxins NAA, 3 indoleacetic acid(IAA) and indolebutyric acid (IBA) were ini-tially assayed, for their effectiveness. Based onthese assays, kinetin and NAA were utilized ex-clusively for all studies. NAA and kinetin wereevaluated at concentrations of l 0. 2.0. 4.0 and8.0 mg/L each in a 4 x 4 factorial study. Thiswas followed by a 5 x 4 factorial study whichincluded NAA at concentrations of 0.0, 0.1, 0.5,1.0 and 2.0 mglL and kinetin at concentrationsof 0. l, 0.5, 1.0 and 2.0 mglL.MS inorganic macronutrient salts at half(0.5 x ), three quarters (0.75 x ) and full strength(1.0 X ), and sucrose at concentrations of 15, 30,and 45 glL were evaluated in a 3 x 3 factorialstudy. Concentrations of TC agar of 0 (using afilter bridge), 4, 8 and 12 mglL were also eval-uated. Five selected cones of the ostrich fernwere evaluated for growth and multiplication invitro using the final medium described in TableIn vitro Plantlet DevelopmentThe basal medium for plantlet developmentstudies unless altered for study purposes, in-cluded: MS macronutrient salts, 0.5 x : MS mi-cronutrient salts; and, in milligrams per litre i-inositol, 100; thiamine.HCl, 0.4; sucrose, 7500;and TC agar, 8000.Forty shoots per treatment were used for allstudies. These were placed, 10 shoots to a 300-mL ointment jar with 50 mL of medium and cov-ered by a double layer saran film. Cultures weremaintained in continuous light at 3000 lx at 25+ l'C for 4 or 6 wk. Plantlets were then eval-uated for fresh weisht. the number of fronds with

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three or more pinna, and the number of rootsgreater than 5 mm in length. LSD..Sucrose levels of 0.0, 7.5, 15.0, 30.0 and 45.0g/L; MS macronutrient levels of 0.125 x ,0.25 x , 0.5 x and 1.0 x the orininal concentra- 6 sootions and TC agar levels of 0 (using a filter 100

bridge), 4, 6, 8, 10, and 12 glL werc evaluated d :::sepaiately in randomized complete block design g :::for their influence on plantlet developmentFive clones were evaluated for their response Fto the selected plantlet development medium in-dicated in Table 5.Post Culture EstablishmentThe move from in vitro culture to soil was bestachieved by moving the culture vessels to ashaded greenhouse for 1 wk prior to transplant-ing. Plants were then transferred to a soiiiess mixin 4-cm cell packs and kept in a high-humiditychamber for 2 wk. They were then moved to aregular shaded greenhouse bench. After 2 mothey were transplanted to 7.5-cm peat pots.

RESULTSShoot MultiplicationPHvroHonvroNE LEVELS. In the initialstudy, NAA had no effect on shoot multi-plication, and increasing levels of NAAsignificantly inhibited culture growth (Fig.1) as expressed by fresh weight. Rates ofkinetin higher than 2 mg/L also inhibitedgrowth and reduced shoot proliferation.There was no significant difference be-tween 1 and 2 mglL of kinetin. There wasa significant interaction between auxin andcytokinin in culture growth. At the highlevels of kinetin, NAA had no effect onfresh weight, but at low kinetin concentra-tions, growth was depressed by increasingNAA rates (Fig. l).In the second study, in which relativelylow concentrations of NAA and kinetinwere tested, NAA had no stimulatory effecton culture growth or shoot multiplication(Table 1), regardless of kinetin concentra-tions. As in the initial experiments, multi-plication was greatest at kinetin rates of Iand 2 mglL. Culture fresh weight decreasedwith increasing kinetin concentrations;therefore individual shoot weight dramati-cally decreased with increasing kinetin

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Fig. l. Influence of growth regulators on in vitrogrowth and shoot multiplication of the ostrichfern (clone NB44).

levels. There was no interaction betweenNAA and kinetin at the low levels tested inthis second study.NurntrtoN AND MEDIUM srRUC-ruRE. Half strength MS macronutrientsalts produced the greatest fresh and dryweights (Table 2). Shoot multiplication wasgreatest with 0.5 x salts while individualshoot weights were greatest at0.15 x. Su-crose levels did not affect multiplicationrates but strongly influenced culture freshweight (Table 2). The 30-glL sucrose treat-ment yielded the greatest fresh weight, butdry weight did not differ between 30 and 45g/L of sucrose. There was no interaction be-tween inorganic salt and sucrose levels inthe medium. Increasing agar levels did notsignificantly reduce either fresh weight or

DYKEMAN AND CUMMING IN VITRO PROPAGATION OF THE OSTRICH FERN

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10213 cANADIAN JouRNAL oF pLANT sctENCETable l. Response of ostrich fern (clone NB44) tissue to levels of NAA and kinetin in culture

NAA(mg/L) Kinetin(mg/L)Freshweight(mg)

Shootsperculture

Calculated D.yweight per weightshoot (mg) (mg)0.00.10.51.02.0

L)l2452t62252162112552391902I

33.930.232.929.028.61 4.-530.241 .937.0

8.3NSNS

7.08.16.67.8/.o

18.78.45.75.1

0.1t,. -)1.02.0

3l3126212830302925

6.8SD (P-0 05)NAAKinetinNAA x kinetin

Signi.ficance (ANOVA)NS NS

NSNS

*xSignificant at l%c level, NS, not significant.Table 2. lnfluence of inorganic salts and sucrose on in vitro shoot proliferation and growth of ostrich fern (cloneNB44) trssue

MS macronutrientsalts Sucrose(concentration) (mg/L)Culturefreshweight

Shootsperc ul ture

Culture Fresh shootdry weightweight (mg)0.50 x0.75 x1.00 x

2t.3I 1.38.8

l2.715 .713.08.9

56.149.438.440.852.051.112.1

30448.631 .036.531 .635.9t2.9

l5,00030,00045,000

516)l I216439543396102SD (P:0 0s)

MS saltsSucroseSalts x Sucrose

SigniJit'ance (ANOVA)

NS NSNSNS

NSNS

x+Significant at rhe l7a level: NS, not significant.the number of shoots per culture. The rateof 4 glL of TC agar produced good growthand shoot multiplication.

When the optimum medium presented inTable 5 was evaluated using five differentclones, there were large differences in re-sponse between clones, but growth ap-peared to be healthy for all clones (TableShoot MorphogenesisObservations indicated that shoots aroseadventitiously, largely on the adaxial sur-

face ofthe developing rachis. Occasionallyshoots would arise from leaf apical andmarginal meristems. When phytohormoneswere present in the medium, leaves under-went little or no lamina development andfrond extension but remained as tight cro-ziers in compact, multiple shoots resem-bling miniature dormant crowns of the spe-cies or leaf buds of higher plants (Fig. 2a).When placed on phytohormone-free me-dium. croziers unfurled without notable de-lay followed by rapid frond growth and rootdevelopment (Fig. 2b). On a medium con-

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DYKEMAN AND CUMMINC - IN VITRO PROPAGATION OF THE OSTRICH FERN IO29Table 3. Clonal responses to in vitro shoot multiplication and plantlet development media

Shoot multiplicaton mediumClone Shoots percul ture Freshweight (mg) Fronds perculture Roots percultureNB5NB I2NB I4NB35NB39P lant Le t dev e Io pme nt medi um

Clone

12.5 + 2.1*22.3 + 4 .6118+l I4.8+1.416 t+3 IFreshweight(mgl

369 + 47225 +24194 + l5334 + 60351 +64Frondsperplantlet

ll0+2.1ll.2:x1.2'7.0+0.26.7 i 0.8t2.6 + 3.5Rootsperplantlet

2.0 + 0.40.00.7 + 0.11.7 +0.'74.1 +2.5No. shootaplces perplantlet

NB5NB I2NB I4NB35NB39

366 + 30214 + 25t36 + 2l188 + l9440 + 30

8.2 + 0.53.2+0.43.1 + 3.031+3.07.9 + 0.5

9.',l +3.03.6+2.74 8+3 57 .8):2 8

13.2 + 5.0

2.1 + 0.92.0 + 1.0| .6 + 0.'71.6+0 63.0 + 0.9

*Mean and standard error of 30 cultures per clone.taining kinetin, each newly initiated leafdeveloped a bud on the adaxial surface ofits rachis. Thus, when macroscopically sin-gle shoots from kinetin-treated cultureswere placed on kinetin free medium, all in-itiated buds developed resulting in a plan-tlet with several shoot apices.Plantlet DevelopmentBecause of the lack of frond and root de-velopment in the presence of phytohor-

mones, a plantlet development stage wasrequired. Plantlet development was very re-sponsive to inorganic nutrients, carbohy-drate, and agar levels in the medium (Table4). NAA had no positive effect on plantletdevelopment. MS macronutrient salts werefinally used at half-strength because rootdevelopment was greatly inhibited at fullstrength.Sucrose levels of 7.5 glL resulted in thegreatest fresh weight and the highest num-

Fig. 2. In vitro culture of the ostrich fern on (a) shoot multiplication medium (3 x ), and (b) plantletdevelopment medium (2 x ).

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1030Table 4. lnfluence

Addendum

CANADIAN JOURNAL OF PLANT SCIENCEof addenda levels on plantlet development and growth of ostrich fern (clone NB44) shootsin a pretransplant medlum

Fresh weight(mgJ

No. developedfronds No. roots)5mmMS macronutrient salt concentration1.0 x0.5 x0.25 x0. 125 xSignificanceLSD (P:0.05)Sucrose (glL)0.01s15.030.0450Significance (ANOVA)LSD (P:0.05)TC agar (glL)0

468l0t2Significance (ANOVA)LSD (P:0.05)

NAA (metL)000.1Significance (ANOVA)LSD (P:0.01)

3534062511336924329

2'751345l44

14818912087785342

208l1545

| 4.613.9I1.58.128/1l3.lt2 l883.08.43.12.82.21.8I -:)0.90.8

4.411.29.0243.00.1129tt.42.'70.3t.23.4403.53lJ.Z2-6NS'7.42.91.0

9.08.3NS**,*Significant at P=0.01 and P:0.05, respectively; NS, not significantber of roots per plantlet when evaluatedafter 4 wk (Table 4). The largest number ofdeveloped fronds was attained with 7.5 and15 .0 g/L of sucrose. If plantlets were left inculture for 6 wk, the l5-g/L treatmentyielded larger plantlets which were darkergreen.Agar concentrations of 0 and 4 g/L in themedium facilitated the greatest growth andfrond development. Each level above 4 glL progressively depressed fresh weight,frond height and the number of developedfronds. Agar concentration had no effect onrootlet development.Various clones responded differently tothe plantlet development medium but use-able plantlets were produced for all clonestested (Table 3).

DISCUSSIONIt was observed that new shoots arose ad-ventitiously on the adaxial surface of thedeveloping crozier rachis. Hoffmeister( I 857) reported the existence of buds on theadaxial surface of the rachis which were laiddown early in the ontogeny of the leaf ofthe ostrich fern. He found that this bud didnot develop beyond a few initials and wassoon overgrown by marginal tissue. Hoff-meister's work suggested that the morpho-genic potential of these initials was lost veryearly in the development of the leaf. In thepresence of kinetin in vitro, these initialsappear to continue developing to producenew shoots.In vitro propagation studies on other fernspecies have consistently reported optimum

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DYKEMAN AND CUMMING - IN VITRO PROPAGATION OF THE OSTRICH FERN 1031Table 5. Proposed media for in vitro propagation ofthe ostrich fern (per litre)Explant establishnrent and shoot multiplicationMS macronutrient saltsMS micronutrient saltsSodrum monobasic phosphateAdenine sulphatei-inositolT'hiamine.HClKinetinSucroseTC agarpH to 5.8Plantlet developmentMS macronutrient saltsMS micronutrient saltsi-inositolThiamine.HClSucroseAgarpH to 5.8

0.5 x0.5 xR5 mo40 mg100 mg0.4 mg1.0 mg30 000 mg4000 mg

0.5 x0.5 x100 mg0.4 mg7500 mg4000 mg

kinetin levels of l-2 mglL (Harper 1976;Beck 1980) but optimum levels of auxinshave varied from 0.1 to 10.0 mg/L of NAA.In the studies reported here, greatest shootmultiplication was also obtained with 1-2mgil of kinetin. Nevertheless, the freshweight of cultures increased with decreas-ing kinetin levels and much larger individ-ual shoots were produced at kinetin levelsbelow I mg/L. No advantage was found inthe addition of NAA to the medium at anyconcentration in these studies.The levels of inorganic salts, sucrose andagar all significantly affected growth, mul-tiplication and morphogenesis. 'fhe normalMS inorganic nutrient levels were too highfor both the multiplication and plant devel-opment stages for this species. Shoot mul-tiplication was most sensitive to inorganicsalt levels.The response to sucrose levels differedmarkedly with the developmental process.While shoot multiplication rate was not sig-nificantly affected by sucrose concentra-tion. culture growth was very sensitive toconcentration. During shoot multplication(an 8-wk passage) maximum growth wasachieved at 30 glL sucrose (osmotic poten-tial (rfn) : - 0.33 mPa) with growth being

significantly less at higher sucrose levels.During plantlet development (a 4-wk pas-sage) maximum growth was attained withonly 7.5 g/L sucrose ( : - 0. 17 mPa).When plantlet developmeni cultures wereleft for 6 wk, 15.0 gil of sucrose (rfn':-0.23 mPa) was superior to 7.5 g/L. Thissuggests that a fine line exists between ad-equate carbohydrate nutrition and the in-hibitory effect of high sucrose concentra-tions (and low osmotic potentials of themedium) on plant growth and morphogenicdevelopment. There have been many ref-

erences in the literature to the effects ofcar-bohydrate levels on callus growth and or-ganogenesis (Thorpe 1982), but relativelyfew referring to morphogenic development.Most reports have concluded that high car-bohydrate levels and/or low medium os-motic potentials are most conducive to or-ganogenesis (Brown and Thorpe 1980), theoptimum level being about 37o (wtivol) formany species (Upper et al. 1970; Barg andUmiel 1976; Aitken et al. 1981). Con-versely, plantlet development of some spe-cies has been depressed at high carbohy-drate levels with optimum levels rangingfrom 0.5 to 2Vo (Al Talib and Torry 1959;Barg and Umiel 1976; Aitken et al. 1981;Cheng and Vogui 1911).The differences inresponse obtained in the present studiescould be explained in part by the differ-ences in duration of the shoot multiplicationand plantlet development passages. It islikely however, that osmotic potential of themedium also plays a role - with the pro-cess of shoot organogenesls requiring ortolerating lower medium osmotic potentialthan the process of morphogenic develop-ment.Plantlets obtained by utilizing the mediadescribed here have been successfullytransplanted to soil and grown to a size suf-ficient for field establishment. To date.l0 000 in vitro cultured plants have beensuccessfully transplanted to soil. Tissue-culture-derived plants are currently beingevaluated for clonal integrity and field per-formance.

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1032 CANADIAN ]OURNAL OF PLANT SCIENCEACKNOWLEDGMENTS

Appreciation is extended to Ms. Terri Brodie forher valuable technical assistance. This work wassupported by the New Brunswick Department ofAgriculture and Rural Development; also a grantin aid of research from the Natural Sciences andEngineering Research Council of Canada to B.G. Cumming is gratefully acknowledged.Aitken, J., Hargan, J. and Thorpe, T. A.1981. Influence of explant selection on shoot-forming capacity of juvenile tissue of Pinus ra-diara. Can. J. For. Res. ll: 112-ll7.Al-Talib, K. H. and Torrey, J. H. 1959. Theaseptic culture of isolated buds of PseudotsugataxifoLia. Plant Physiol. 34: 630-637.Barg, R. and Umiel, N. 1977. Effects of sugarconcentration on growth, greening and shootformation in callus cultures from four geneticlines of tobacco. Z. pflanzenphysiol. 81: 153-160.Beck, M. J. f980. The effects of kinetin andnaphthaleneacetic acid on in vitro shoot multi-plication and rooting in the fishtail fern. M.Sc.thesis. University of Tennessee, Knoxville, Ten.31 pp.Brown, D. C. W. and Thorpe, T. A. 1980.Changes in water potential and its componentsduring shoot formation in tobacco callus. Phys-iol. Plant 49: 83-87.

Harper, K. L. 1976. Asexual multipiication ofleptosporangiate ferns through tissue culture.M.S. thesis. University of California, River-side, Calif.Cheng, T. Y. and Vogui, T. H' 1977. Regen-eration of Douglas Fir plantlets through tissueculture. Science 198: 306-307.Hoffmeister, W. 1857. Beitrage zur kenntnisder Geffaskryptogamen. Abh. Kon. Sachs Ges.Wiss. 3: 603-682.Hughes, K. W. 1981. Ornamental species.Pages 5-50 in B. V. Conger, ed. Cloning agri-cultural plants. CRC Press, Boca Raton, Fla.Murashige, T. and Skoog, T. 1962, A revisedmedium for rapid growth and bioassays with to-bacco tissue culture. Physiol. Plant. 15: 473-49'7 .Thorpe, T. A. 1982. Carbohydrate utilizationand metabolism. Pages 325-268 in J. M. Bongaand D. J. Durzan, eds. Tissue culture in for-estry. Nijhoff and Junk, The Hague, The Neth-erlands.Upper, C. D., Helgeson, J. P. and Haberlach'G. T. 1970. Limitations of tobacco callus, tis-sue growth by carbohydrate availability. PlantPhysiol. 46: ll8-122.Wardlaw, C. W. 1943. Experimental and an-alytical studies of pteridophytes. I. Preliminaryobservations in the development of buds on rhi-zomes of the ostrich fern (Matteuccia struthiop-terls Tod.). Ann. Bot. N.S. 7: l'71-184.p

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Ostrich Purin

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